Methods For Targeted Complementary DNA Enrichment

Abstract

The present invention provides methods for enriching a target complementary DNA (cDNA), comprising: (a) providing a plurality of cDNAs, each comprising a first universal sequence at an end, and wherein the plurality of cDNAs comprises the target cDNA; (b) amplifying the target cDNA with a universal forward primer complementary to the first universal sequence and a gene specific reverse primer, and wherein a second universal sequence is added to an end of the cDNA opposite the first universal sequence, by a nucleic acid amplification reaction, by ligation, or by a primer extension reaction; and (c) amplifying the amplicons or extension products using the universal forward primer and a universal reverse primer complementary to the second universal sequence. In one embodiment, the universal forward primer, the gene specific reverse primer and the second universal reverse primer are provided in the same reaction mixture such that the amplifying is a single step.

What is claimed is: 
     
         1 . A method of enriching a target complementary DNA (cDNA), comprising the steps of:
 (a) providing a plurality of different cDNAs, wherein each cDNA comprises a first universal sequence at an end, and wherein the plurality of cDNAs comprises the target cDNA to be enriched;   (b) providing a first reaction mixture comprising:
 1) a first universal oligonucleotide forward primer comprising a sequence that is complementary to all or a portion of the first universal sequence, and 
 2) at least one gene specific reverse primer, wherein the at least one gene specific reverse primer comprises a sequence that is complementary to all or a portion of a sequence in the target cDNA, and further comprises at least one second universal sequence at an end of the at least one gene specific primer; 
   (c) contacting the plurality of cDNAs with the first reaction mixture;   (d) amplifying the target cDNA to obtain an amplicon;   (e) providing a second reaction mixture comprising:
 1) a second universal oligonucleotide forward primer comprising a sequence that is complementary to all or a portion of the first universal sequence, and 
 2) a universal oligonucleotide reverse primer, wherein the universal oligonucleotide reverse primer comprises a sequence that is complementary to all or a portion of the at least one second universal sequence; thereby enriching for the target cDNA; 
   (f) contacting the amplicon with the second reaction mixture; and   (g) amplifying the amplicon, thereby enriching for the target cDNA.   
     
     
         2 . The method of  claim 1 , wherein each cDNA in the plurality of cDNAs further comprises a cell identification tag or a unique molecular identifier (UMI) sequence, or a combination thereof. 
     
     
         3 . The method of  claim 2 , wherein the cell identification tag or UMI sequence, or combination thereof, further comprises a poly(T) sequence. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the first universal sequence is at the 5′ end of each cDNA molecule. 
     
     
         5 . The method of any one of  claims 1 - 3 , wherein the first universal sequence is at the 3′ end of each cDNA molecule. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein the plurality of cDNAs is obtained by reverse transcribing mRNA from a single cell. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein amplifying the target cDNA in (d) comprises amplifying by polymerase chain reaction. 
     
     
         8 . The method of any one of  claims 1 - 6 , wherein the at least one gene specific reverse primer further comprises a blocking domain separated from the primer by an RNA base. 
     
     
         9 . The method of  claim 8 , wherein amplifying the target cDNA in (d) comprises amplifying by an RNase H-dependent polymerase chain reaction. 
     
     
         10 . The method of any one of  claims 2 - 9 , wherein the cell identification tag or the UMI sequence, or the combination thereof, are preserved after the target cDNA is amplified. 
     
     
         11 . The method of any one of  claims 1 - 10 , further comprising sequencing one or more portions of the target cDNA using the universal oligonucleotide forward primer, the universal oligonucleotide reverse primer, the at least one gene specific reverse primer, or a combination thereof. 
     
     
         12 . A method of enriching a target complementary DNA (cDNA), comprising the steps of:
 (a) providing a plurality of cDNAs, wherein each cDNA comprises a first universal sequence at an end, and wherein the plurality of cDNAs comprises the target cDNA to be enriched;   (b) providing a first reaction mixture comprising:
 1) a universal oligonucleotide forward primer comprising a sequence that is complementary to all or a portion of the first universal sequence, and 
 2) a gene specific reverse primer comprising a sequence that is complementary to a sequence in the target cDNA; 
   (c) contacting the plurality of cDNAs with the first reaction mixture;   (d) amplifying the target cDNA to obtain a first amplicon;   (e) adding at least one second universal sequence to an end of each target cDNA in the first amplicon to obtain a conjugated amplicon;   (f) providing a second reaction mixture comprising:
 1) a universal oligonucleotide forward primer comprising a sequence that is complementary to all or a portion of the first universal sequence, 
 2) a universal oligonucleotide reverse primer comprising a sequence that is complementary to all or a portion of the at least one second universal sequence; 
   (g) contacting the conjugated amplicon with the second reaction mixture; and   (h) amplifying the conjugated amplicon, thereby enriching for the target cDNA.   
     
     
         13 . The method of  claim 12 , wherein each cDNA in the plurality of cDNAs further comprises a cell identification tag or a unique molecular identifier (UMI) sequence, or a combination thereof. 
     
     
         14 . The method of  claim 13 , wherein the cell identification tag or UMI sequence, or combination thereof, further comprises a poly(T) sequence. 
     
     
         15 . The method of any one of  claims 12 - 14 , wherein the first universal sequence is at the 5′ end of each cDNA molecule. 
     
     
         16 . The method of any one of  claims 12 - 14 , wherein the first universal sequence is at the 3′ end of each cDNA molecule. 
     
     
         17 . The method of any one of  claims 12 - 16 , wherein the plurality of cDNAs is obtained by reverse transcribing mRNA from a single cell. 
     
     
         18 . The method of any one of  claims 12 - 17 , wherein at least one of the amplifying steps comprises amplifying by polymerase chain reaction. 
     
     
         19 . The method of any one of  claims 12 - 17 , wherein the gene specific reverse primer further comprises a blocking domain separated from the primer by an RNA base. 
     
     
         20 . The method of  claim 19 , wherein at least one of the amplifying steps comprises amplifying by an RNase H-dependent polymerase chain reaction. 
     
     
         21 . The method of  claim 12 , wherein the at least one second universal sequence is ligated to an end of the first amplicon opposite the first universal sequence. 
     
     
         22 . The method of  claim 12 , wherein one end of the universal oligonucleotide forward primer has a blocking group, thereby blocking phosphorylation of the universal oligonucleotide forward primer on the one end. 
     
     
         23 . The method of  claim 22 , wherein the blocking group is an inverted dTTP. 
     
     
         24 . The method of any one of  claims 22 - 23 , wherein the blocking group is at the 5′ end of the universal oligonucleotide forward primer. 
     
     
         25 . The method of  claim 12 , wherein the at least one second universal sequence is added using transposons. 
     
     
         26 . The method of  claim 12 , wherein the at least one second universal sequence is added by ligating the at least one second universal sequence to an end of each target cDNA in the first amplicon, by a primer extension reaction, or by a nucleic acid amplification reaction, or a combination thereof. 
     
     
         27 . The method of  claim 12 , wherein the at least one second universal sequence is added by fragmenting nucleic acid molecules in the first amplicon and ligating the at least one second universal sequence to the fragments, by a primer extension reaction, or by a nucleic acid amplification reaction, or a combination thereof. 
     
     
         28 . The method of  claim 12 , wherein the at least one second universal sequence is added at an end opposite to the at least one first universal sequence. 
     
     
         29 . The method of any one of  claims 12 - 28 , wherein the cell identification tag or the UMI sequence, or the combination thereof, are preserved after the conjugated amplicon of target cDNA is amplified. 
     
     
         30 . The method of any one of  claims 12 - 29 , further comprising sequencing one or more portion of the target cDNA using the universal oligonucleotide forward primer, the universal oligonucleotide reverse primer, the gene specific reverse primer, a gene specific forward primer, or a combination thereof. 
     
     
         31 . A method of enriching a target complementary DNA (cDNA), comprising the steps of:
 (a) providing a plurality of cDNAs, wherein each cDNA comprises a first universal sequence at an end and wherein the plurality of cDNAs comprises the target cDNA to be enriched;   (b) providing a first reaction mixture comprising:
 at least one gene specific primer comprising a sequence that is complementary to all or a portion of a sequence in the target cDNA, and further comprises at least one second universal sequence at an end of the at least one gene specific primer; 
   (c) contacting the plurality of cDNAs with the first reaction mixture such that the at least one gene specific primer hybridizes with the target cDNA;   (d) extending the at least one gene specific primer to obtain at least one extension product;   (e) providing a second reaction mixture comprising:
 1) a universal oligonucleotide forward primer comprising a sequence that is complementary to all or a portion of the first universal sequence; and 
 2) a universal oligonucleotide reverse primer comprising a sequence that is complementary to all or a portion of the at least one second universal sequence; 
   (f) contacting the at least one extension product with the second reaction mixture; and   (g) amplifying the at least one extension product, thereby enriching for the target cDNA.   
     
     
         32 . The method of  claim 31 , wherein each cDNA in the plurality of cDNAs further comprises a cell identification tag or a unique molecular identifier (UMI) sequence, or a combination thereof. 
     
     
         33 . The method of  claim 32 , wherein the cell identification tag or UMI sequence, or combination thereof, further comprises a poly(T) sequence. 
     
     
         34 . The method of any one of  claims 31 - 33 , wherein the first universal sequence is at the 5′ end of each cDNA molecule. 
     
     
         35 . The method of any one of  claims 31 - 33 , wherein the first universal sequence is at the 3′ end of each cDNA molecule. 
     
     
         36 . The method of any one of  claims 31 - 35 , wherein the plurality of cDNAs is obtained by reverse transcribing mRNA from a single cell. 
     
     
         37 . The method of any one of  claims 31 - 36 , wherein amplifying the at least one extension product in (g) comprises amplifying by polymerase chain reaction. 
     
     
         38 . The method of any one of  claims 31 - 37 , wherein the cell identification tag or the UMI sequence, or the combination thereof, are preserved after the at least one extension product is amplified. 
     
     
         39 . The method of any one of  claims 31 - 38 , further comprising sequencing one or more portion of the target cDNA using the universal oligonucleotide forward primer, the universal oligonucleotide reverse primer, the gene specific primer, or a combination thereof. 
     
     
         40 . The method of any one of  claims 31 - 39 , wherein the first reaction mixture comprises a polymerase deficient in flap endonuclease activity. 
     
     
         41 . The method of any one of the preceding claims, further comprising the step of sequencing the enriched target cDNA. 
     
     
         42 . A method of enriching a target complementary DNA (cDNA), comprising the steps of:
 (a) providing a plurality of different cDNAs, wherein each cDNA comprises a first universal sequence at an end, and wherein the plurality of cDNAs comprises the target cDNA to be enriched;   (b) providing a reaction mixture comprising:
 1) a universal oligonucleotide forward primer comprising a sequence that is complementary to all or a portion of the first universal sequence, 
 2) at least one gene specific reverse primer, wherein the at least one gene specific reverse primer comprises a sequence that is complementary to all or a portion of a sequence in the target cDNA, and further comprises at least one second universal sequence at an end of the at least one gene specific primer, and 
 3) a universal oligonucleotide reverse primer, wherein the universal oligonucleotide reverse primer comprises a sequence that is complementary to all or a portion of the at least one second universal sequence; 
   (c) contacting the plurality of cDNAs with the reaction mixture; and   (d) amplifying the target cDNA, thereby enriching for the target cDNA.   
     
     
         43 . The method of  claim 42 , wherein each cDNA in the plurality of cDNAs further comprises a cell identification tag or a unique molecular identifier (UMI) sequence, or a combination thereof. 
     
     
         44 . The method of  claim 43 , wherein the cell identification tag or UMI sequence, or combination thereof, further comprises a poly(T) sequence. 
     
     
         45 . The method of any one of  claims 42 - 44 , wherein the first universal sequence is at the 5′ end of each cDNA molecule. 
     
     
         46 . The method of any one of  claims 42 - 44 , wherein the first universal sequence is at the 3′ end of each cDNA molecule. 
     
     
         47 . The method of any one of  claims 42 - 46 , wherein the plurality of cDNAs is obtained by reverse transcribing mRNA from a single cell. 
     
     
         48 . The method of any one of  claims 42 - 47 , wherein amplifying the target cDNA in (d) comprises amplifying by polymerase chain reaction. 
     
     
         49 . The method of any one of  claims 42 - 47 , wherein the at least one gene specific reverse primer further comprises a blocking domain separated from the primer by an RNA base. 
     
     
         50 . The method of  claim 49 , wherein amplifying the target cDNA in (d) comprises amplifying by an RNase H-dependent polymerase chain reaction. 
     
     
         51 . The method of any one of  claims 43 - 50 , wherein the cell identification tag or the UMI sequence, or the combination thereof, are preserved after the target cDNA is amplified. 
     
     
         52 . The method of any one of  claims 42 - 51 , further comprising sequencing one or more portions of the target cDNA using the universal oligonucleotide forward primer, the universal oligonucleotide reverse primer, the at least one gene specific reverse primer, or a combination thereof.